Abstract
BackgroundUnlike its constitutive isoforms, including neuronal and endothelial nitric oxide synthase, inducible nitric oxide synthase (iNOS) along with a series of cytokines are generated in inflammatory pathologic conditions in retinal photoreceptors. In this study, we constructed transgenic mice overexpressing iNOS in the retina to evaluate the effect of sustained, intense iNOS generation in the photoreceptor damage.MethodsFor construction of opsin/iNOS transgene in the CMVSport 6 expression vector, the 4.4 kb Acc65I/Xhol mouse rod opsin promoter was ligated upstream to a 4.1 kb fragment encoding the complete mouse cDNA of iNOS. From the four founders identified, two heterozygote lines and one homozygote line were established. The presence of iNOS in the retina was confirmed and the pathologic role of iNOS was assessed by detecting nitrotyrosine products and apoptosis. Commercial TUNEL kit was used to detect DNA strand breaks, a later step in a sequence of morphologic changes of apoptosis process.ResultsThe insertion and translation of iNOS gene were demonstrated by an intense single 130 kDa band in Western blot and specific immunolocalization at the photoreceptors of the retina. Cellular toxicity in the retinas of transgenic animals was detected by a post-translational modification product, tyrosine-nitrated protein, the most significant one of which was nitrated cytochrome c. Following the accumulation of nitrated mitochondrial proteins and cytochrome c release, marked apoptosis was detected in the photoreceptor cell nuclei of the retina.ConclusionsWe have generated a pathologic phenotype with sustained iNOS overexpression and, therefore, high output of nitric oxide. Under basal conditions, such overexpression of iNOS causes marked mitochondrial cytochrome c nitration and release and subsequent photoreceptor apoptosis in the retina. Therefore, the modulation of pathways leading to iNOS generation or its effective neutralization can be of significant therapeutic benefit in the oxidative stress-mediated retinal degeneration, a leading cause of blindness.
Highlights
There are three known isoforms of nitric oxide synthase and all three isoforms generate nitric oxide (NO) by the catalytic conversion of arginine to citrulline [1]
An excessive amount of NO generation [3] by inducible nitric oxide synthase (iNOS) in the pathologic conditions is elicited by immune system activators, such as endotoxins and the cytokines, including interleukin-I (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) [4,5,6]
Deletion of iNOS gene prevented oxidative stress and simultaneously abrogated the peroxynitrite-mediated tyrosine nitration in the retinal photoreceptors in experimental autoimmune uveoretinitis (EAU). While these results suggest a causative role of iNOS in retinal pathology, the specific contribution of upregulated iNOS expression isolated from that of inflammatory cytokines was not evaluated [10]
Summary
There are three known isoforms of nitric oxide synthase and all three isoforms generate nitric oxide (NO) by the catalytic conversion of arginine to citrulline [1]. Endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) are restricted to the defined subcellular domains and require calcium and calmodulin for their activation. These two isoforms are constitutively present to generate a small amount of NO for physiological functions [1], [2]. Once produced rapidly scavenges the superoxide to form the potent biological oxidant peroxynitrite, which is known to cause irreversible tissue damage [7] With this known detrimental potential, iNOS-toxicity has been found in several ocular inflammatory diseases [4] as well as in neuropathological diseases with marked inflammatory components, such as mutiple sclerosis, Parkinson’s disease, and the early stages of Alzheimer’s disease [8]. We constructed transgenic mice overexpressing iNOS in the retina to evaluate the effect of sustained, intense iNOS generation in the photoreceptor damage
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